The objectives of this proposal are to develop a high performance photon counting imager optimized for ultrahigh sensitivity fluorescence spectroscopy and imaging and to develop new methodologies and protocols that take advantage of its unique capabilities. The detector will be capable of registering single photons with high-resolution in space and time at high-count rates and high quantum efficiency. It will be suitable for imaging and spectroscopy of single molecules, molecular complexes and macromolecules in living cells and tissues with increased sensitivity, signal to noise and signal to background, while providing multi-parametric, high information-content from each detected photon. They dub this detector H33D (pronounced `heed', for: high-spatial resolution, high-temporal resolution and high count-rate 3-dimensional detector). The targeted detector performance: (i) a spatial resolution of 100 microm x 100 microm per pixel (spatially resolvable units) with a detector dimension (25 mm diameter) allowing at least 256 x 256 resolution elements (pixels), (ii) 100 picoseconds (ps) temporal resolution, (iii) a maximum count rate of 10(5) Hertz (100 kHz) per single pixel and 5.10(7) Hertz (50 MHz) over the whole detector and (iv) a quantum efficiency (QE) of > 40 percent @ 600nm. The proposed work is "high risk, high impact" and fits well the structure of the R21/R33 phased innovation mechanism. They will develop 3 generations of H33D detectors. The specifications/milestones for the pilot phase are already superior to those of current commercial detectors. They anticipate that these milestones will be met within 12 months. In the expanded development phase (R33), they will build two more generations. To deliver on the milestones, they will need to develop high QE photocathode (feasibility unknown, high risk); low-gain, moderate-resolution spatial detection (proof of principle exists); time-stamping of simultaneous spatially-separable events (feasibility unknown, high risk); high-count rate at high timing resolution (feasibility good). The development of the H33D would open new windows in multi-parameters studies of single molecules (simultaneous acquisition of lifetime, fluorescence spectrum and polarization), in time-resolved wide-field imaging of in vivo cellular processes or in vitro enzymatic reactions and high-throughput fluorescence correlation spectroscopy (FCS). They will demonstrate proof-of-principle biological applications building on other funded projects in their laboratory: (i) Time-correlated imaging and spectroscopy of semiconductor nanocrystals (NCs); (ii) Digital time-gated imaging of NCs in live fibroblast cells; (iii) Fluorescence lifetime imaging (FLIM) of live cells; (iv) Line-confocal time-resolved spectral imaging of cells; (v) Single-pair FRET conformational dynamics of surface-immobilized RNA polymerase molecules; (vi) Microfluidic applications; (vii) Multiconfocal fluorescence correlation spectroscopy (FCS); (viii) High frame-rate cell imaging.